Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease

Abstract

Objective. To identify and characterize a novel autoantibody, anti-WS, that binds total transfer RNA (tRNA).Methods. Serum from patient WS, who had polyarthritis, Sjogren's syndrome, Raynaud's phenomenon, and interstitial pulmonary fibrosis, was used in this study. Characteristics of anti-WS and antibody-reactive determinants of tRNA were investigated by 32P immunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro.Results. WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence. 32Pimmunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8s and 5sribosomal RNAs from 32P-labeled HeLa cell extract. When deproteinized RNA was used as antigen source, total tRNAs were still precipitated by WS serum. An immunoprecipitation study, using various deletion mutants of Escherichiu coli tRNA, demonstrated that both D and T I / J C loops were needed for antibody binding.Substitution of nucleotide lSC with 18A of E coli tRNAIrW, which is essential in the formation of the tertiary "L" shape of tRNA, inhibited binding by anti-WS antibodies. Conclusion. Anti-WS antibodies are novel autoantibodies directed against tRNAs. The antibody binding site is the common L-shaped tertiary structure conformed by the D loop and T I , ! J

Anti-transfer RNA antibodies in two patients with pulmonary fibrosis, Raynaud's phenomenon and polyarthritis

“…While some patients also produce autoantibodies directed against naked RNAs [1][2][3][4][5], the antigenic determinants of the RNA and the clinical presentation of patients possessing such autoantibodies have not been investigated extensively. We have previously described novel autoantibodies that recognise naked transfer RNAs (tRNAs) [6]. We have found another patient who has anti-tRNA antibodies with the same specificity and we describe here the clinical characteristics of these patients.…”

“…Immunoprecipitation was performed as described previously [6,7]. Approximately 2 x 107 HeLa cells were cultured at a cell density of 2 x 105 cells/ml for 12 h. Harvested cells were sonicated and the cell supernatant was used as antigen.…”

Inositol 1,4,5-trisphosphate receptors are autoantibody target antigens in patients with Sjögren's syndrome and other systemic rheumatic diseases

“…13 Anti-SS-A/Ro, SS-B/La, U1RNP, Sm, Scl 70 (Topo-isomerase 1), and anti-mitochondrial antibodies were further confi rmed by enzyme-liked immunosorbent assay using commercial kits. Immunoprecipitation was used for the confi rmation of anti-Ki, Ku, 14 rRNP, Wa, 15 WS, 16 and p97/VCP. 8 Immunoblotting for anti-IP 3 Rs antibodies Microsome fractions were prepared from Sf9 cells overexpressing mouse IP 3 Rs as described previously.…”

Inosine and N 1-methylinosine within a synthetic oligomer mimicking the anticodon loop of human tRNAAla are major epitopes for anti-PL-12 myositis autoantibodies

“…Several autoantibodies that recognize various deproteinized RNAs have been described (reviewed in Hoet & Van Venrooij, 1992;Keene, 1996;Mimori, 1996)+ Among them, only those against U1-U5 snRNA, as detected in few patients with systemic sclerosis, were shown to be directed to a specific modified nucleoside (2,2,7-trimethylguanosine cap structure) (Okano & -methylinosinic acid (pm 1 I) are higher in F than in E because the ratio between modified and nonmodified nucleotides in the variant RNA 7 is higher than for variant RNA 5+ The solvent system 1 as described in Auxilien et al+ (1996) was used in all cases+ Medsger, 1992)+ Except for the autoantibodies against tRNA Ala (anticodon IGC) described in the present study, the other anti-RNA autoantibodies characterized so far are not directed to modified nucleotides, as they also immunoprecipitate in vitro transcripts, lacking modified nucleosides, of the natural or synthetic genes corresponding to these antigenic RNAs+ Examples include autoantibodies against a characteristic 40-nt stem-loop fragment in U1snRNA (Deutscher & Keene, 1988;Tsai et al+, 1992;Teunissen et al+, 1998), a 59-nt fragment of 28S rRNA (Chu et al+, 1991), a 84-nt fragment of Y5 Ro RNA (Boulanger et al+, 1995), or various mutants of tRNA retaining their L-shaped three-dimensional architecture (Matsumura et al+, 1996;Brouwer et al+, 1998;Ohosone et al+, 1998)+ In these cases a few selected canonical nucleotides within a well-defined RNA conformation (a characteristic stem-loop or a three-dimensional architecture) constitute the major epitopes recognized by the corresponding IgG+ It is worth mentioning that antibodies predominantly directed towards the characteristic hypermodified nucleoside wybutosine, which is located at position 37 of the anticodon loop of yeast tRNA Phe , were obtained by immunizing a goat with a glutaraldehyde-mediated conjugate of yeast tRNA Phe and bovine gamma globulin (Fuchs et al+, 1974)+ Also, antibodies against a variety of modified nucleosides, including inosine (Inouye et al+, 1971) and 1-methylinosine (D'Ambrosio et al+, 1991; Renaud et al+, 1991) have been prepared by immunizing rabbits, mice or goats with a conjugate of the hapten and a protein carrier (reviewed in Vold, 1990)+ In this article, we present evidence that the important features recognized by human anti-tRNA Ala (anti-PL-12) autoantibodies include the chemical identity and the spatial disposition of two modified ribonucleotides within the anticodon loop of tRNA Ala (anticodon IGC): namely the bases inosine at position 34 and N 1 -methylinosine at position 37 (see Fig+ 1A), their spacing, and probably their three-dimensional configuration+ Figure 5 shows the spatial orientation of the 6-keto groups (in black) of inosine-34 and of the N 1 -methyl group (in grey) of N 1 -methylinosi...…”

“…Sera from patients with the chronic, inflammatory muscle disorder myositis (including polymyositis and dermatomyositis), commonly contain autoantibodies directed against one or more components (autoantigens) of RNA or protein biosynthetic pathways (reviewed in Plotz et al+, 1995;Mimori, 1996)+ The most common myositisspecific autoantibodies, seen in about one third of patients, are directed against the cytoplasmic aminoacyltRNA synthetases (antisynthetase syndrome)+ Each of the 20 aminoacyl-tRNA synthetases catalyzes the charging of several cognate tRNA species with specific amino acid+ To date, autoantibodies have been characterized against several aminoacyl-tRNA synthetases, including those specific for histidine (Jo-1), threonine (PL-7), alanine (PL-12), isoleucine (OJ and NJ), glycine (EJ), asparagine (KS), lysine, and tryptophane (reviewed in Targoff, 1992Targoff, , 1994, see also Paley et al+, 1995;Gelpi et al+, 1996;Hirakata et al+, 1999, and references therein)+ Within this group, anti-histidyl-tRNA synthe-tase (anti-Jo-1) is the most frequently found, occurring in about 20% of myositis patients (Nishikai & Reichlin, 1980;Vazquez-Abad & Rothfield, 1996)+ Except for anti-PL-12 and anti Jo-1 (see below), these autoantibodies immunoprecipitate the synthetase associated with its cognate tRNAs but the epitopes are located within the protein components (for examples, see Ramsden et al+, 1989;Raben et al+, 1994;Kato et al+, 1995;Ripmaster et al+, 1995;Rutjes et al+, 1997)+ In contrast, sera of patients of the PL-12 type contain separate populations of autoantibodies that react with either alanyl-tRNA synthetase or naturally occurring (fully matured) RNA Ala (Bunn et al+, 1986;Bunn & Mathews, 1987b)+ As a further distinction, the tRNA Ala and alanyltRNA synthetase autoantigens are mutually exclusive and do not coprecipitate with the same autoantibodies (Bunn et al+, 1986)+ Additional evidence that PL-12 myositis sera contain distinct sets of autoantibodies comes from the observation that the relative amounts of immunoglobulin (IgG) against the alanyl-tRNA synthetase and against tRNA Ala vary considerably from one patient to another, and during the course of clinical treatment (S+R+ Brand, Y+ Corda, H+ Grosjean & M+ Mathews, unpubl+ observations)+ A similar situation has recently been discovered with certain patients afflicted with the Jo-1 type of myositis+ In this case, distinct autoantibodies against deproteinized tRNA His and histidyl-tRNA synthetase also coexist in sera (Brouwer et al+, 1998)+…”